Thousands of chemical substances are examined each year for their biotransformation and potential toxicity. Nearly all chemical toxicity tests will be conducted on rats, mice, guinea pigs, rabbits, cats, dogs and monkeys, with the results from these animal experiments being extrapolated to humans. While the reactions of rats, rabbits and mice to chemicals together with metabolite patterns are currently considered acceptable predictors of the toxic effects these substances will have on the human organism, the significance of animal experimentation and subsequent data extrapolation is a questionable science since species variation can be significant. By utilizing human in vitro biotransformation technology, the fate and disposition of potentially toxic, environmental/industrial chemicals or prospective drugs in man can be more accurately determined without exposing living humans or laboratory animals to the actual compounds.
For the past several years, metabolism has been studied using subcellular (microsome) fractions obtained from human livers. Systems such as this are important for metabolite identification and rate of formation. The use of such subcellar fractions only allows one to determine if a particular enzymatic process exists within the fraction and its ability to react with a given compound. However, the observance of a specific enzymatic activity in an isolated subcellular fraction does not mean that this reaction will be expressed to the same extent in vivo. Extensive cellular organization and compartmentalization within the cell determine chemical disposition of a test compound. Thus, it would be optimal to have an in vitro system that retains the tissue's inherent organization and which will best reflect human in vivo biotransformation. An intact in vitro metabolism system would also mimic both the metabolic and chemical reactions that occur during in vivo metabolism.
Intact sections of human tissues appear to maintain crucial characteristics of cellular organization while allowing the researcher the versatility of an in vitro system. This is important since there is a heterogeneous distribution of biotransformation enzymes within the tissue and intact tissue sections sample all of these sites exactly as in the whole organism.
Human livers, kidneys, lungs, hearts and pancreas are harvested from organ donors usually in a hospital or organ bank and made available through a nationwide network the transplant centers. Occasionally, suitable matches can not be found or the prospective recipient develops complications and the tissue then becomes available for research. In order to do certain biological studies such as xenobiotics metabolism, intermediary metabolism, hormone receptor studies or chemical induced toxicity, the tissue has to be fully intact and comparable in its viability to the standards set for transplantable tissue. This optimal viability is particularly important for biochemical and cell biological studies requiring fully functional cells.
Two investigations, Pollard and Dutton, reported limited success using tissue fragments and slices in drug disposition studies, but mentioned two specific problems associated with their methods. The first was their inability to prepare uniform, viable sections rapidly. The second problem centered around the failure to maintain tissue viability throughout the experiment.
In the mid 1980's the first problem encountered by Pollard and Dutton was overcome by the introduction of the Krumdieck et al. tissue slicer to produce non-traumatized, precision-cut liver slices (.+-.5% variation in thickness) from 60 to 500 microns thick at a rapid rate under physiological conditions.
The problem of maintaining tissue viability during an experiment, however, has not yet been satisfactorily overcome. Inappropriately cultured tissue sections no longer have the gas, fluid and nutrient exchange that exist in vivo. To overcome these deficiencies, an incubation system was developed in which the liver slices were exposed to the gas phase while the side resting on the support was constantly bathed in media. The media was supposed to coat the whole slice by capillary action, and the submerged side receives gas via diffusion. This however, proved inadequate to maintain the slice. The submerged slice rapidly deteriorated and only cells in the gas-tissue interface remained viable. A number of investigators have maintained slices in culture for 12 hours to several days with only the cells at the gas-tissue phase remaining viable.
An organ culture system was developed in an attempt to overcome the inadequate oxygenation of the conventional culture system. This new system combined the advantages of increased surface area for oxygenation and nutrient uptake with those of conventional organ culture (maintain organ architecture). Smith et al. floated slices into cylindrical, stainless steel screen supports fitted with two narrow stainless steel sleeves. The tissue adhered to the screens during culture. The cylinders were then placed inside vials that contain sufficient media to wet the slices when the vials are on their sides. The vials were flushed with 95:5% O.sub.2 :CO.sub.2, sealed, and incubated on a heated (37.degree. C.) vial rotator. As the vial was rotated the support inside also rotated causing the underside of the tissue to be alternatively exposed to media or gas while the upper side was continuously exposed to gas. This dynamic organ culture system was far superior to conventional systems resulting in a completely viable slice for up to 48 hours. In addition, it has been reported that animal liver slices seemed to improve during culturing, raising the possibility that the human liver slices might actually repair themselves during this type of culturing.
U.S. Pat. No. 4,920,044 to Bretan describes an intracellular flush solution for preserving organs. By way of background, Bretan provides the compositions of four other flush solutions, namely: Collins-2 Flush; Sacks-2 Flush; Belzer perfusate; and UW-1 Flush. Bretan states that D-glucose has been recently shown to exacerbate acute renal ischemia damage in dogs. Mannitol is therefore substituted for glucose in the Bretan solution (Bretan, column 10, lines 32-39).
U.S. Pat. No. 4,798,824 to Belzer et al. describes perfusate for the preservation of organs, particularly kidneys. The Belzer et al. '824 perfusate contains hydroxyethyl starch ("HES") in place of human serum albumin for colloidal osmotic support.
U.S. Pat. No. 4,879,283 to Belzer et al. describes a solution which includes hydroxyethyl starch ("HES") for the preservation of organs. Belzer et al. indicate that glucose, the main impermeant in Collins' solution, is not an effective impermeant for the liver or pancreas and readily enters cells. Thus, Belzer et al. completely remove glucose from their formulation and replace glucose with HES.
Belzer et al. have also reported that preservation solutions containing high concentrations of glucose pose a two-fold disadvantage since glucose is not an effective impermeant to prevent cell swelling and glucose could stimulate acidosis. Belzer et al., Transplantation, Vol. 45, pgs. 673-676, (April, 1988).
No prior cold preservation solution known to applicants has been successful in maintaining the viability of tissue for longer than 72 hours.
It is an object of this invention to provide a preservation solution which permits the cold preservation of viable tissue for periods longer than three days, preferably for seven to ten days.
It is a further object of this invention to provide a method of cold preserving tissue for up to ten days while maintaining viability of the tissue and for cryopreserving tissue for extended periods up to three years while maintaining the viability of the tissue.
It is also an object of this invention to expand the availability of human tissue for testing, including xenobiotics metabolism, intermediary metabolism, hormone receptor and chemical induced toxicology testing.
It is a further object of this invention to provide human tissue samples for testing, which tissue samples preserve the cellular organization and compartmentalization within the human tissue sample. It is a further object of the present invention to provide an organ culture system which allows for the accurate determination of in vivo biotransformation with an in vitro system.
It is also an object of this invention to provide a method of freezing and storing viable human tissues. It is a further object of this invention to provide an integrated procedure which includes tissue slicing-cold storage-cryopreservation-special packaging for shipping and later culturing-cold storage during transport and packaging in specified self-righting cold storage containers. All steps may advantageously be performed in one basic solution, which is modified by one additional ingredient (DMSO) for cryopreservation and replacement of some potassium for sodium in the final culturing phase. It is also an object of this invention to provide a preservation method which will allow the establishment of a tissue bank comprised of valuable human tissue slices to be utilized for research.
It is a further object of this invention to reduce the amount of preclinical animal trials by providing a method for the preservation and distribution of viable human tissue slices for testing.